The sequence of the rightmost 4,626 bp of the Bacillus phage φ29 genome is presented and analyzed. Nine large open reading frames (ORF's) have been found. Three of these ORF's are correlated with the late genes 14, 15 and 16. The remaining six ORF's are in the right early region. One of these early ORF's has been identified as gene 17 (g17), the only early gene to have been genetically mapped in this region. The remaining ORF's (16.5, 16.6, 16.7, 16.8 and 16.9) were previously unknown. The biological efficacies of some of these putative early ORF's were demonstrated using an in vitro E. coli transcription-translation system. The primary amino acid sequences, molecular weights, translational initiation sequences and genetic organization of these nine genes are presented and discussed. Gene product 15 (gp15) was found to have strong homology with Salmonella phage P22 gp19, a lysozyme. gp15 also has a lesser but possibly significant homology with T4 gene product e (gpe), also a lysozyme. Using a clone containing φ29 g15 it was shown that gp15 can complement T4 gene e (ge) mutant infections, leading to the conclusion that φ29 g15 encodes a lysozyme. Three transcriptional initiation sites (P(E)3, P(EC)3 and B2) were previously mapped in this region. The sequences of the putative P(EC)3 and B2 promoter sites are presented and shown to have homology with the Bacillus σ⁵⁵ concensus sequence. Sequences having homology to a minor Bacillus sigma factor recognition site, σ³², are also presented and discussed. The region between the last late gene (g16) and the last early gene (ORF-16.5) consists of only 30 bp. Analysis of potential secondary structures of transcripts across this region suggests that the same sequences may be involved in the termination of both late and early transcription.

published_or_final_version / Microbiology / Master / Master of Medical Sciences

Thesis (MSc)--University of Stellenbosch, 2000. / ENGLISH ABSTRACT: Esterases play a variety of roles in nearly every aspect of life ranging from cellular metabolism, signal transduction to defence mechanisms in plants. One aspect of esterases that recently is receiving more attention is the role esterases play in the .degradation of plant material. With fossil fuels (coal and oil) estimated to run out in the next 20 to 30 years, renewable sources such as plant biomass are becoming increasingly important. Plant biomass contains hemicellulosic and cellulosic materials that need to be degraded to their different constituents before they can be optimally used for the production of commodities. Although the enzymes needed to hydrolyse the xylan backbone (xylanases and P-xylosidases) are important, enzymes that remove side chains from the polymer are equally important. They facilitate hydrolysis by xylanases and P-xylosidases and will improve the availability of monomeric sugars for utilisation when used in conjunction with other xylanolytic enzymes. Many of these side-chains are esters and they need to be removed through the action of esterases, either directly from the xylan backbone or from shorter xylo-oligomers. An existing genomic DNA library of Bacil/us pumilus in Escherichia coli was screened for the presence of an acetyl esterase encoding gene. Positive clones were identified by the formation of clearing zones on plates containing glucose pentaacetate. Plasmid DNA was isolated from a positive E. coli clone. The DNA insert was sequenced and found to contain two open reading frames, one of which encoded a novel esterase (estA). Using different primers the gene was amplified by polymerase chain reaction and inserted into an inducible expression vector (pKK223- 3) for expression in E. coli. The plasmid was introduced into E coli and the esterase activity determined, using the chromogenic substrate a-naphthyl acetate. Activity levels decreased shortly after induction with IPTG and therefore plasmid pAP4 was used for enzymatic assays. Cultures containing plasmid pAP4 produced extracellular activity of 2.5 nkatal/ml. The pH and temperature optima as well as temperature stability of the enzyme was determined. The enzyme exhibited optimal activity at pH 6 and 60°C and was stable at 60°C after 2 h. Enzyme assays on different substrates yielded activity on methylumbelliferyl butyrate and methylumbelliferyl acetate in addition to the glucose pentaacetate and a-naphthyl acetate. The estA gene was cloned into a yeast expression vector between the PGK promoter and terminator sequences for expression of the gene in Saccharomyces cerevisiae. The estA open reading frame was also fused to the MFa 1 secretion signal for secretion of the protein from S. cerevisiae. The expression vector was successfully transformed into S. cerevisiae, but no extracellular activity was detected. Only low intracellular activity of 0.260 nkatal/ml was detected in S. cerevisiae. / AFRIKAANSE OPSOMMING: Esterases speel 'n verskeidenheid van rolle in feitlik elke aspek van lewe, van sel metabolisme, sein transduksie tot verdedigingsmeganismes in plante. Een aspek van esterases wat al hoe meer aandag geniet, is die rol wat esterases in die afbraak van plant en plantmateriaal speel. Met olie- en steenkoolbronne wat na beraming oor 20 tot 30 jaar tot niet sal gaan, raak die rol wat hernubare bronne speel al hoe belangriker. Plantbiomassa bevat sellulose en hemisellulose wat tot die verskillende komponente afgebreek moet word voordat dit optimaal vir die vervaardiging van produkte aangewend kan word. Alhoewel die ensieme wat vir die hidrolise van die xilaanruggraat benodig word, (xilanases en ~-xulosidases) belangrik is, is die ensieme wat die sygroepe vanaf die polimeer verwyder ewe belangrik aangesien hulle die hidrolise deur xilanases en ~-xulosidases bevorder. Wanneer hulle saam met die ander xilanolitiese ensieme gebruik word, sal hulle die beskikbaarheid van monomeriese suikers vir fermentasie verhoog. Baie van hierdie sygroepe is esters en hulle word deur die aksie van esterases verwyder, of direk van die ruggraat, ofvanafkorter xilo-oligosakkariede. 'n Bestaande genoom DNA biblioteek van Bacillus pumilus in Escherichia coli is vir die teenwoordigheid van 'n asetielesterase-koderende geen gesif. Positiewe klone is deur die vorming van 'n sone op plate wat glukose pentaasetaat bevat, geïdentifiseer. Die DNA-invoeging van die positiewe E. coli-kloon se DNA-volgorde is bepaal en twee oopleesrame is gevind waarvan een vir 'n unieke esterase (estA) kodeer. Met behulp van verskillende inleiers is die geen met die polimerasekettingreaksie (PKR) geamplifiseer en in 'n induseerbare promotor vir uitdrukking in E. coli gekloneer. Die plasmied is getransformeer in E. coli en aktiwiteit is bepaal deur cc-naftielasetaatte gebruik. Vlakke van aktiwiteit het kort na induksie met IPTG weer gedaal en daarom was plasmied pAP4 vir ensiematiese toetse gebruik. E. coli-transformante met plasmied pAP4 het ekstrasellulêre aktiwiteit van 2.5 nkatal/ml gelewer. Die pH en temperatuur optima sowel as die temperatuurstabiliteit van die ensiem was bepaal. Die ensiem toon optimale aktiwiteit by pH 6 en 'n temperatuur van 60°C. Aktiwiteitstoetse op verskillende substrate het aktiwiteit op metielumbelliferielasetaat en metielumbelliferielbutiraat bo-en-behalwe die glukosepentaasetaat en c-naftielasetaar getoon. Die estA geen is in uitdrukkingskasette bevattende die PGKpromotor en-termineerder vir uitdrukking in Saccharomyces cerevisiae gekloneer. Dit is ook agter die MFal-sekresiesein gekoppel vir sekresie vanuit S. cerevisiae. Geen ekstrasellulêre aktiwiteit is gevind nie. Slegs intrasellulêre aktiwiteit van 0.26 nanokatal per mililiter was bepaal.

Graduation date: 1994

Thesis (MScAgric)--University of Stellenbosch, 2002. / ENGLISH ABSTRACT: Cellulose, a ~-1,4-linked polymer of glucose, is the most abundant renewable carbon source on earth. It is well established that efficient degradation of cellulose requires the synergistic action of three categories of enzymes: endoglucanases (EG), cellobiohydrolases (CBH) and ~-glucosidases. ~-Glucosidases are a heterogenous group of enzymes that display broad substrate specificity with respect to hydrolysis of cellobiose and different aryl- and alkyl-ê-u-glucosides. They not only catalyse the final step in the saccharification of cellulose, but also stimulate the extent of cellulose hydrolysis by relieving the cellobiose mediated inhibition of EG and CBH. The ability to utilize cellobiose is widespread among gram-negative, gram-positive, and Archaea bacterial genera. Cellobiose phosphoenolpyruvate- dependent phosphotransferase systems (PTS) have been reported in various bacteria, including: Bacillus species. In this study, we have used a cellobiose chromophore analog, p-nitrophenyl- ~-D-glucopyranoside (pNPG), to screen a Bacillus pumilus genomic library for cellobiose utilization genes that are functionally expressed in Escherichia coli. Cloning and sequencing of the most active clone with subsequent sequence analysis allowed the identification of four adjacent open reading frames. An operon of four genes (celBACH), encoding a cellobiose phosphotransferase system (PTS): enzyme II (encoded by celB, celA and celC) and a ó-phospho-f-glucosidase (encoded by celH) was derived from the sequence data. The amino acid sequence of the celH gene displayed good homology with ~-glucosidases from Bacillus halodurans (74.2%), B. subtilis (72.7%) and Listeria monocytogenes (62.2%). .As implied by sequence alignments, the celH gene product belongs to family 1 of the glycosyl hydrolases, which employ a retaining mechanism of enzymatic bond hydrolysis. In vivo PTS activity assays concluded that the optimal temperature and pH at which the recombinant E. coli strain hydrolysed pNPG were pH 7.5 and 45°C, respectively. Unfortunately, at 45°C the CelBACH-associated activity of the recombinant strain was only stable for 20 minutes. It was also shown that the enzyme complex is very sensitive to glucose. Since active growing cells metabolise glucose very rapidly this feature is not a significant problem. Constitutive expression of the B. pumilus celBACH genes in E. coli enabled the host to efficiently metabolise cellobiose as a carbon source. However, cellobiose utilization was only achievable in the presence ofO.01% glucose. This phenomenon could be explained by the critical role of phosphoenolpyruvate (PEP) as the phosphate donor in PTS-mediated transport. Glucose supplementation induced the glycolytic pathway and subsequently the availability of PEP. Furthermore, it could be concluded that the general PTS components . (enzyme I and HPr) of E. coli must have complemented the CelBACH system from B. pumilus to allow functionality of the celBACH operon, in the recombinant E. coli host. / AFRIKAANSE OPSOMMING: Sellulose (' n polimeer van p-l,4-gekoppelde glukose) is die volopste bron van hernubare koostof in die natuur. Effektiewe afbraak van sellulose word deur die sinnergistiese werking van drie ensiernklasse bewerkstellig: endoglukanases (EG), sellobiohidrolases (CBH) en P-glukosidases. p-Glukosidases behoort tot 'n heterogene groep ensieme met 'n wye substraatspesifisiteit m.b.t. sellobiose en verskeie ariel- and alkiel-ê-n-glukosidiesc verbindings. Alhoewel hierdie ensieme primêr as kataliste vir die omskakeling van sellulose afbraak-produkte funksioneer, stimuleer hulle ook die mate waartoe sellulose hidroliese plaasvind deur eindprodukinhibisie van EG en CBH op te hef. Sellobiose word algemeen deur verskeie genera van die gram-negatiewe, gram-positiewe en Archae bakterieë gemetaboliseer. Die sellobiose-spesifieke fosfoenolpirovaatfosfotransportsisteem (PTS) is reeds is in verskeie bakterië, insluitende die Bacillus spesies, beskryf. In hierdie studie word die sifting van 'n Bacillus pumilus genoombiblioteek m.b.V. 'n chromofoor analoog van sellobiose, p-nitrofeniel-p-o-glukopiranosied (pNPG), vir die teenwoordigheid van gene wat moontlike sellobiose-benutting in Escherichia coli kan bewerkstellig, beskryf. Die DNA-volgorde van die mees aktiewe kloon is bepaal en daaropvolgende analiese van die DNA-volgorde het vier aangrensende oopleesrame geïdentifiseer. 'n Operon (celBACH), bestaande uit vier gene, wat onderskeidelik vir die ensiem II (gekodeer deur celB, celA en celC) en fosfo-B-glukosidase (gekodeer deur celH) van die sellobiose-spesifieke PTS van B. pumilus kodeer, is vanaf die DNA-volgorde afgelei. Die aminosuuropeenvolging van die celH-geen het goeie homologie met P-glukosidases van Bacillus halodurans (74.2%), B. subtilis (72.7%) en Listeria monocytogenes (62.2%) getoon. Belyning van die DNA-volgordes het aangedui dat die celH geenproduk saam met die familie 1 glikosielhidrolases gegroepeer kan word. Hierdie familie gebruik 'n hidrolitiese meganisme waartydens die stoigiometriese posisie van die anomeriese koolstof behou word. PTS-aktiwiteit van die rekombinante E. coli ras, wat die celBACH gene uitdruk, is in vivo bepaal. Die optimale temperatuur en pH waarby die rekombinante ras pNPG hidroliseer, is onderskeidelik pH 7.5 en 45°C. Alhoewel die ensiernkompleks baie sensitief is vir glukose, is dit nie 'n wesenlike probleem nie, omdat aktief groeiende E. coli selle glukose teen 'n baie vinnige tempo benut. Die celBACH operon het onder beheer van 'n konstitiewe promotor in E coli die rekombinante gasheer in staat gestelom sellobiose as 'n koolstofbron te benut. Die benutting van sellobiose word egter aan die teenwoordigheid van 'n lae konsentrasie glukose (0.01 %) gekoppel. Hierdie verskynsel dui op die kritiese rol van fosfoenolpirovaat (PEP) as die fosfaatdonor gedurende PTS-gebaseerde transport. Glukose speel waarskynlik 'n rol in die indusering van glikoliese, en sodoende die produksie van PEP as tussenproduk. Verder kan afgelei word dat die algemene PTS komponente (ensiem I en HPr) van E. coli die B. pumilis CelBACH-sisteem komplementeer en derhalwe funksionering van die celBACH operon in E. coli toelaat.

Bacillus cereus is traditionally thought to be the only member of its genus accepted as a pathogen in foods like grains, fruits, vegetables and milk due to the presence of the nonhemolytic (Nhe) operon. However, many other Bacillus spp. may also harbor the Nhe operon and be pathogenic. Real-time PCR targeted the nheA gene in 37 samples obtained from food, soil, and reference cultures by analyzing the standard deviations of melt peaks. Rep-PCR was used to compare the banding patterns of each sample against B. cereus ATCC14579 and three B. thuringiensis strains to “fingerprint” each isolate. Of the original 43 isolated tested, 37 were Gram-positive rods. The remaining six samples were Gram-positive cocci. Twenty-five of the 37 Gram-positive Bacillus spp. were nheA positive, while twelve were negative. Many of the nheA positive strains were species not previously known to contain Nhe, and were capable of causing gastroenteritis in consumers. / Department of Biology